Direct Sequencing of PCR Products

It is quite possible to directly sequence a PCR product without first cloning the
fragment. Indeed, there are some distinct advantages to this approach. However, you
need to be aware of some of the drawbacks as well. Direct PCR sequencing is rarely
successful unless you spend some time ensuring that you aren't falling into one of
the many traps. This document will explain how to get sequence directly from a PCR
product with a reasonable chance of success.

Make SURE you amplified the right fragment.

You would be amazed at how often a PCR reactions SEEMS to produce the correct band,
when in fact it has amplified something spurious. Sometimes when you sequence the band,
you will discover that the sequence is completely unexpected and nonsensical. At
other times, sequencing with one of your PCR primers will give a completely blank lane,
while the other primer
will give two simultaneous and superimposed (and thus unreadable) sequences. That happens
when only one of your primers acted as *both* ends of an illegitimate amplification.

Make sure you really are amplifying the fragment you expected. For example, if you know
of a restriction site in the fragment, try to cut it and look for the correct product
bands. Alternatively, use nested primers to re-amplify the desired product to verify
its identity and coincidentally eliminate any illegitimate products.

You must remove all residual PCR primers and unincorporated nucleotides.

Sequencing uses one primer, while PCR utilizes two. If we try to sequence with two
primers present, you'll get the two sequences back, superimposed on each other and
completely unreadable.

There are many ways to purify a PCR reaction prior to sequencing it. Several manufacturers
make kits to do this task. We prefer to not recommend specific products, but ask your
neighbors for their advice. Some people just gel-elute the PCR band, which not only
removes the extraneous primers and nucleotides, but also eliminates illegitimate PCR
products (see below).

If the PCR primers will also be the sequencing primer(s), make sure they match our conditions.

You may be able to adjust your PCR conditions to optimize reactions, but we unfortunately
cannot do this. Please make sure your primer(s) are appropriately designed for automated
sequencing. They should have a Tm between 60 and 70 degrees (or at least between 55 and 75
at the outside), and should have little propensity for primer-dimer formation. Click
here
for complete information on primer design for DNA sequencing.

PCR fragments are smaller, and thus more effective sequencing templates than are the
usual plasmids. Consequently they don't need to be at as high a concentration. For
example, a 200 bp fragment only needs to be 1 ng/ul! If your samples are at too
high a concentration, not only will they NOT sequence any better, they may cause
problems for other peoples' samples nearby. PLEASE estimate your PCR product
concentrations on an analytical gel, and dilute them according to our recommendations
(please see the page
DNA Concentrations for Automated Sequencing.

We may refuse samples from clients who routinely submit overly-concentrated templates.

Inefficient primers are sometimes OK for PCR, but the same primers may fail in sequencing.

Because PCR is intrinsically an exponential process, and because it is usually carried
well beyond completion, even rather poor primers will produce amplification in a PCR
reaction. Sequencing. however, is strictly linear, and is much more unforgiving of poor
primers.

If you have to cycle more than 35 or so times to get an amplification product, or if you
have to use unusual additives or odd conditions to achieve success, your primer may not
be efficient enough to use for sequencing.

If your primer is mismatched to your *original* template, after the PCR reaction, the
product (which of course now incorporates your primers) will indeed match the primers
perfectly. In other words, mismatch primers aren't a problem *if* they're the ones
with which you amplified.

Please DON'T try to use a spectrophotometer to measure the concentration of a PCR product!

Typical laboratory spectrophotometers cannot with any accuracy measure the small amount of
DNA that a PCR reaction generates. Unless you own one of the newer micro-specs (e.g. "Nanodrop"
or similar), you should simply use an analytical agarose gel to estimate the concentration
of your templates. Please compare them to a reference DNA fragment of similar size and known
concentration!

PCR Reactions RARELY produce only single-bands.

You may think that your reaction produced just a single product, but there are very
often other things there. When you use an agarose gel to assess the PCR result, you
can't detect any of these:

Problem: Small, illegitimate products

Amplification at an illegitimate site that gives rise to a small (<100 nt) fragment
will never show up on the typical 1% agarose gel, but it will sequence much better
that your larger, 'legitimate' product.

Solution: Sequence it anyway. If you see interfering peaks for the first 20-100 nt
(especially if they are extremely large), you should assume there's interference
from a small amplification product. If you get the sequence you needed despite the
problem, great. If not, you can always cut the desired band out of a preparative gel,
or go back and redesign your PCR reaction to avoid that interfering product. You may
need to use a 2-3% agarose gel to see smaller fragments reliably.

Warning, however: those small products can produce such bright bands that they cause
interference with someone else's samples nearby! If we complain about your 'overconcentrated'
samples, please consider whether the small fragments are causing problems, and please don't
keep sending such samples if they are!

Problem: A diffuse, low-level background of illegitimate products

When you assess the outcome of a PCR reaction, look closely at your analytical gel for
a dim background smear of ethidium bromide staining. If it's there, it's probably DNA,
and (because it's distrubuted and diffuse) it's probably a LOT of DNA in many PCR bands.
It could easily comprise the majority of DNA in your sample, and the sequencing result
could be very bad.

Solution: well, you could sequence it anyway; sequencing is very cheap these days. If
you do see multiple superimposed peaks instead of clean sequence, you need to clean up
your product some more. Cut the desired band out of a preparative gel, or go back and
redesign your PCR reaction to avoid that interfering product.

Problem: Your "single band" is really two superimposed products

It's surprising, but this is not a particularly rare event. Everything looks great,
but when you sequence it, all you see is multiple, superimposed sequences.This may
occur if you are amplifying two related - but non-identical - genes, or if you have
a homopolymer tract (e.g. poly-A or poly-T that causes the polymerase to 'stutter',
or simply that you were unlucky.

Solution: If you have some way to PROVE that you amplified only the right product, it
is always worth the effort to do so. Don't just assume that the right size means the right
band. For instance, if you happen to know of a restriction site within your expected
PCR fragment, then cut the PCR product and verify that you get the expected fragment(s).
If there are unexpected bands, unexpected sizes or bands that do not cut, be supicious
of the identity or purity of your product.

The use of nested PCR primers can minimize this problem. It is less likely that an
illegitimate product will co-amplify through a second round of PCR with internally-
nested primers.

Sounds a bit dismal, right? Not necessarily. There are some very good reasons
you might want to go ahead and sequence directly from a PCR product. Here are some:

Direct sequencing is much quicker.

If you're screening hundreds of patient samples for mutations in a gene, you do NOT
want to be gel-purifying all those PCR reactions, and you CERTAINLY don't want to
clone them all before sequencing.

Direct sequencing doesn't show any PCR mutations.

Common PCR protocols (Taq polymerase under standard cycling coonditions) generate
mis-incorporations occasionally (about once per 3 kb, in my hands). If you clone
those PCR products and sequence several of them, you will see point mutations
in some of the clones.

If you directly sequence the PCR product, though, what you'll see is the consensus base
at each position. Although many of the individual products have mutated nucleotides,
these mutations are scattered randomly, and are different for each individual product
fragment. Consequently, at any one nucleotide, most of the clones will be correct, and
you'll be seeing the original sequence with no mutations.

We have many clients who have successfully sequenced thousands - even tens of thousands -
of PCR products, with outstanding results. Be critical of the quality of your PCR product
and, if necessary, optimize the PCR conditions or gel-purify the desired fragment. Prove
you have the right fragment before you invest in large-scale sequencing, and you'll be
pleased with the results.